Time-resolved fluorometry PCR assay for rapid detection of herpes simplex virus in cerebrospinal fluid

被引:38
作者
Hukkanen, V
Rehn, T
Kajander, R
Sjöroos, M
Waris, M
机构
[1] Univ Turku, Dept Virol, FIN-20520 Turku, Finland
[2] Univ Turku, MediCity Res Lab, FIN-20520 Turku, Finland
[3] Univ Turku, Dept Biotechnol, FIN-20520 Turku, Finland
[4] Univ Turku, Biophys Lab, FIN-20520 Turku, Finland
关键词
D O I
10.1128/JCM.38.9.3214-3218.2000
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
We have introduced a time-resolved fluorometry (TRF)-based microwell hybridization assay for PCR products in detection of herpes simplex virus (HSV) in cerebrospinal fluid (CSF) specimens. TRF is a sensitive nonradioactive detection technique which involves the use of lanthanide chelates as fluorescent labels. We used PCR primers from the glycoprotein D genes of HSV type 1 (HSV-1) and HSV-2. The biotinylated PCR products were collected on streptavidin-coated microtitration wells and hybridized with short oligonucleotide probes, europium labeled for HSV-1 and samarium labeled for HSV-2, The TRF results were obtained as counts per second and as signal-to-noise (S/N) ratios. The sensitivity of the assay was 0.1 infectious units (PFU) of HSV in CSF specimens, and the S/N values increased with the virus amount, up to 68.5 for 10(3) PFU of HSV-1 and to 58.5 for 10(3) PFU of HSV-2, allowing semiquantitation of HSV in CSF. The primers and probes recognized all the studied 48 HSV wild-type samples, with S/N ratios of 12.4 to 190 (HSV-1) and 5.1 to 248 (HSV-2). We tested CSF specimens, 100 for each HSV type, which were HSV PCR negative by Southern blot and 22 CSF specimens which were HSV-1 or -2 PCR blot positive. In the TRF test, the mean S/N ratio for the HSV-1-negative CSF was 1.37 (standard deviation [SD] = 0.513) and for the HSV-2-negative CSF it was 1.03 (SD = 0.098), The HSV-1 blot-positive CSF yielded S/N ratios of 3.6 to 85.9, and the HSV-2 blot-positive CSF yielded ratios from 1.9 to 13, Using the mean S/N ratio for negative CSF specimens + 3 SD as the cutoff yielded all the previously HSV-positive specimens as TRF positive. The TRF PCR assay for HSV in CSF specimens is a rapid and sensitive method, improves interpretation of PCR results, and is well suited for automation.
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页码:3214 / 3218
页数:5
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